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Team:Chiba/Assay/uptake
From 2013.igem.org
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<h2>uptake実験方法</h2> | <h2>uptake実験方法</h2> | ||
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| + | <br><b>Expreriment:</b><br> | ||
| + | <br>Fig.1,Fig.2に示すプラスミドでそれぞれ形質転換したBL21株について、アラビノースおよびatC存在下で培養し、それぞれフェリチンの発現またはfur, fieFのノックダウンを誘導した後、クエン酸鉄存在下で培養し、呈色試薬による吸光度(Abs756)から大腸菌に取り込まれず培養液中に残存した鉄の濃度を測定した(Fig.3)。 | ||
| + | <br><br><b>Detailed procedure</b> | ||
| + | <br> | ||
1.E.coli strain BL21 were transformed with the plasmids shown in Fig. 1 and Fig.2.<br> | 1.E.coli strain BL21 were transformed with the plasmids shown in Fig. 1 and Fig.2.<br> | ||
2.All transformants were inoculated into culture(100 mL TB medium) and was shaken for 12h (at 37℃).<br> | 2.All transformants were inoculated into culture(100 mL TB medium) and was shaken for 12h (at 37℃).<br> | ||
Revision as of 05:09, 27 September 2013
uptake実験方法
Expreriment:
Fig.1,Fig.2に示すプラスミドでそれぞれ形質転換したBL21株について、アラビノースおよびatC存在下で培養し、それぞれフェリチンの発現またはfur, fieFのノックダウンを誘導した後、クエン酸鉄存在下で培養し、呈色試薬による吸光度(Abs756)から大腸菌に取り込まれず培養液中に残存した鉄の濃度を測定した(Fig.3)。
Detailed procedure
1.E.coli strain BL21 were transformed with the plasmids shown in Fig. 1 and Fig.2.
2.All transformants were inoculated into culture(100 mL TB medium) and was shaken for 12h (at 37℃).
3.We centrifuged samples with 3,750 rpm, at 4℃ for 25 minutes and resuspended with 5 mL LB medium.
4.We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37℃).
5.We diluted samples by LB medium and got 500 uL of the diluted cell suspension. Approximately, 2×10^9, 10^9, 5×10^8 cells were contained in it.
6.We added 5 uL from 10 mM iron citrate(Ⅲ) and 5uL from 10 mM iron ascorbate(Ⅱ) to the sample, and we shake the sample for 1h (at 37℃) to charge iron inside E.coli.
7.We centrifuged the sample with 19,600 rpm, at 37℃ for 1 minutes and got supernatant(60 mL).
8.We added color reagent, Tris buffer solution, Reducing Agent to the sample and measured absorbance (Abs756) to determine the density of iron.
絵とかあるといいよね！！
